Compositions and methods comprising long-chain, straight-chain 2-amino-3-hydroxyalkanes

ABSTRACT

Investigation of the activity of extracts of the clam  Spisula polynyma  has led to antitumor long-chain, straight-chain alkane or alkene compounds which have a 2-amino group and a 3-hydroxy group. The present invention is directed to compositions and methods comprising an isolated and purified long-chain, straight-chain 2-amino-3-hydroxyalkane, or prodrug thereof, and a pharmaceutically acceptable carrier, wherein the carbon chain in the long-chain, straight-chain 2-amino-3-hydroxyalkane is C 16 –C 24 .

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of application Ser. No. 09/386,724,filed Aug. 31, 1999, allowed as U.S. Pat. No. 6,800,661, which is acontinuation-in-part of application Ser. No. 09/058,456, filed Apr. 10,1998, allowed as U.S. Pat. No. 6,107,520, re-issued as U.S. RE38,793,which claims the benefit of U.S. Provisional Application Ser. No.60/043,326, filed Apr. 15, 1997, and U.S. Provisional Application Ser.No. 60/043,599, filed Apr. 15, 1997. The disclosure of application Ser.No. 09/058,456 is incorporated herein by reference. Also incorporatedherein by reference are the disclosures of application Ser. Nos.60/043,326 and 60/043,599 which are themselves incorporated by referencein application Ser. No. 09/058,456.

This invention was made with Government support under Contract NumberAI-04769 awarded by the Naional Institutes of Health (NIH). TheGovernment has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to pharmaceutical compositions ofspisulosine compounds. It further relates to the treatment of tumors,and provides new cytotoxic compounds and pharmaceutical compositions foruse against tumors. In one aspect, the invention relates to antitumorcompounds from marine organisms.

BACKGROUND OF THE INVENTION

There has been considerable interest in isolating bioactive compoundsfrom marine organisms. Typical procedures involve in vitro screeningprograms to test crude extracts for antimicrobial, antiviral, andcytotoxic activities. Illustrative examples of known bioactive compoundsfrom marine sources include bryostatins, ecteinascidins and furthermoredidemnins where didemnin B, also now known as aplidine, is the firstmarine natural product in clinical testing.

SUMMARY OF THE INVENTION

The present invention provides new pharmaceutical compositionscontaining a long-chain, straight-chain alkane or alkene compound whichhas a 2-amino group and a 3-hydroxy group, together with apharmaceutically acceptable carrier. Typically the compound is a2-amino-3-hydroxyalkane or a 2-amino-1,3-dihydroxyalkene. Preferably thecompound is a substituted C₁₆–C₂₄ alkane or alkene. The compound ispreferably a substituted alkane, more preferably a substituted C₁₈–C₂₀alkane, and most preferably a 2-amino-3-hydroxy C₁₈ alkane. Thesubstituted alkene is preferably a substituted mono- or di-alkene, morepreferably a substituted C₁₈–C₂₀ alkene. In one embodiment, thecompounds have the partial stereochemistry:

In particular, the present invention provides compositions which containbioactive sphingoid-type bases, spisulosines 285, 299 and 313 (1–3),sphingosine (also referred to as 4-sphingenine oroctadeca-4-sphingenine, 4) and two related compounds,nonadeca-4-sphingenine (a one carbon longer homologue, 5) andsphinga-4,10-diene (a dehydrosphingosine derivative, 6).

Thus, the preferred compositions contain one or more of the followingpreferred compounds:

spisulosine 285 (1), n=12; spisulosine 299 (2), n=13; spisulosine 313(3), n=14;

as well as sphingosine (4), n=12 and nonadeca-4-sphingenine (5), n=13;and

sphinga-4,10-diene (6).

The preferred compound, spisulosine 285, is known in the literature.Compound 1 and the syn diastereoisomer, were first synthesized byCroatian researchers in the determination of absolute configurations oflipid bases with two or more asymmetric carbon atoms, see Pro{hacek over(s)}tenik, M., Alaupovic, P. Croat. Chem. Acta. 1957, 29, 393.

It is believed that the other compounds in the compositions of thisinvention are new compounds.

Compounds 1–3 show unique cytotoxicity against L1210 murine lymphocyticleukemia cells. In a number of the L1210 assays, a distinctmorphological alteration was observed. This effect was also described inour earlier U.S. Provisional Patent Application Ser. No. 60/043,326. Wemake no patent claim in this patent application to the effect itself onL1210, and indeed there is now some preliminary data that suggests thatthe compounds such as spisulosine 285 might lack activity againstleukemia tumors.

A synthetic sample of 1 was assayed against L1210 leukemia cells andshowed both cytotoxicity and morphological alteration, pointed cellactivity.

L1210 Inhibition and pointed cell activity Concentration % cytotoxicity% pointed cells^(a)  0.5 μg/ml 100 97 0.25 μg/ml 99 100  0.1 μg/ml 99 620.05 μg/ml 96 71 0.025 μg/ml  90 21 0.01 μg/ml 45 1 ^(a)Percent pointedcells are a percent of the living cells.

Spisulosine 285 (1) is also active against other tumor cell lines invitro, including P-388 (0.01 mg/ml); A-549 (0.05 mg/ml); HT-29 (0.05mg/ml) and MEL-28 (0.05 mg/ml).

In a particularly preferred embodiment, the present invention relates touse of spisulosine 285, and related compounds, in the treatment of alltypes of cancer, such as breast cancers, prostate, bladder, pancreas,lung, esophagus, larynx, liver, colon, thyroid, melanoma, kidney,testicular, leukemia, ovarian, gastro-intestinal, hepatocellularcarcinoma and vascular endothelial cancer. Other forms of cancer arewell known to the person skilled in the art. It is preferred that theuse of spisulosine 285, and related compounds is against solid tumors,with use against slow proliferating tumors such as prostate, lung,liver, kidney, endocrine gland and vascular endothelial cancerparticularly preferred. In one aspect, the compositions are for use intherapy directed at the vascular endothelium for control of tissue andtumor vascularisation.

The present invention is directed to bioactive compounds that have beenfound to possess specific antitumor activities and as such they will beuseful as medicinal agents in mammals, particularly in humans. Thus,another aspect of the present invention concerns pharmaceuticalcompositions containing the active compounds identified herein andmethods of treatment employing such pharmaceutical compositions.

The active compounds of the present invention exhibit antitumoractivity. Thus, the present invention also provides a method of treatingany mammal affected by a malignant tumor sensitive to these compounds,which comprises administering to the affected individual atherapeutically effective amount of an active compound or mixture ofcompounds, or pharmaceutical compositions thereof. The present inventionalso relates to pharmaceutical preparations, which contain as activeingredient one or more of the compounds of this invention, as well asthe processes for its preparation.

Examples of pharmaceutical compositions include any solid (tablets,pills, capsules, granules, etc.) or liquid (solutions, suspensions oremulsions) with suitable composition or oral, topical or parenteraladministration, and they may contain the pure compound or in combinationwith any carrier or other pharmacologically active compounds. Thesecompositions may need to be sterile when administered parenterally.

Administration of the composition of the present invention may be by anysuitable method, such as intravenous infusion, oral preparations,intraperitoneal and intravenous administration. Intravenous delivery maybe carried out over any suitable time period, such as 1 to 4 hours oreven longer if required, at suitable intervals of say 2 to 4 weeks.Pharmaceutical compositions containing spisulosine may be delivered byliposome or nanosphere encapsulation, in sustained release formulationsor by other standard delivery means.

The correct dosage of a pharmaceutical composition comprising thecompounds of this invention will vary according to the particularformulation, the mode of application, and the particular situs, host andbacteria or tumor being treated. Other factors like age, body weight,sex, diet, time of administration, rate of excretion, condition of thehost, drug combinations, reaction sensitivities and severity of thedisease shall be taken into account. Administration can be carried outcontinuously or periodically within the maximum tolerated dose.

The compounds may be provided in the pharmaceutical compositions of thisinvention in the form of a prodrug or precursor, which uponadministration converts or is metabolized to the active compound.

The compositions of this invention may be used with other drugs toprovide a combination therapy. The other drugs may form part of the samecomposition, or be provided as a separate composition for administrationat the same time or a different time. The identity of the other drug isnot particularly limited, and suitable candidates include:

a) drugs with antimitotic effects, especially those which targetcytoskeletal elements, including microtubule modulators such as taxanedrugs (such as taxol, paclitaxel, taxotere, docetaxel), podophylotoxinsor vinca alkaloids (vincristine, vinblastine);

b) antimetabolite drugs such as 5-fluorouracil, cytarabine, gemcitabine,purine analogues such as pentostatin, methotrexate);

c) alkylating agents such as nitrogen mustards (such as cyclophosphamideor ifosphamide);

d) drugs which target DNA such as the antracycline drugs adriamycin,doxorubicin, pharmorubicin or epirubicin;

e) drugs which target topoisomerases such as etoposide;

f) hormones and hormone agonists or antagonists such as estrogens,antiestrogens (tamoxifen and related compounds) and androgens,flutamide, leuprorelin, goserelin, cyprotrone or octreotide;

g) drugs which target signal transduction in tumour cells includingantibody derivatives such as herceptin;

h) alkylating drugs such as platinum drugs (cis-platin, carboplatin,oxaliplatin, paraplatin) or nitrosoureas;

i) drugs potentially affecting metastasis of tumours such as matrixmetalloproteinase inhibitors;

j) gene therapy and antisense agents;

k) antibody therapeutics; and

l) other bioactive compounds of marine origin, notably theecteinascidins such as ET-743, or the didemnins such as aplidine.

The present invention also extends to the compounds for use in a methodof treatment, and to the use of the compounds in the preparation of acomposition for treatment of cancer.

Spisulosine 285 has an effect upon cell morphology. Vero cells treatedwith spisulosine 285 had a reduced microfilament structure, as assessedby staining of the spisulosine-treated cells with phalloidin, whichstains actin in the microfilaments. Spisulosine 285 also affects thedistribution of the small GTP binding protein Rho, although this effectmay be reduced or eliminated by pre-treatment with the Rho-activator LPA(Mackay and Hall, J. Biol. Chem., 273, 20685–20688, 1998).

Without wishing to be constrained by theory, we believe that themechanism of action of spisulosine 285 may involve modulation of theaction of the small GTP binding protein Rho, possibly via an effect onLPA activation. Rho is known to be involved in the formation of stressfibers (Hall, A., Science 279, 509–514, 1998), and has a role incontrolling cell adhesion and motility through reorganization of theactin cytoskeleton (Itoh, et al, Nature Medicine, Vol 5, No. 2, 1999).Adhesion of tumor cells to host cell layers and subsequent transcellularmigration are key steps in cancer invasion and metastasis. By affecting(reducing) the levels of microfilaments in the cell, via an (inhibitory)effect on Rho, spisulosine 285 may serve to limit the development ofcancer via an effect on the cell cytoskeleton. It is also known that Rhotriggers progression of the G1 phase of the cell cycle. As such,modulation of Rho may also prevent cellular transformation by stoppingcell cycle progression. Therefore, the present invention also relates tothe use of spisulosine compounds in the preparation of a medicament forthe treatment of cancer, wherein the spisulosine compound acts to alterRho protein activity.

LPA, an activator of Rho, can help prevent the effect of spisulosinecompounds on microfilament formation. While the specific target ofspisulosine 285 is not known, the observed reduction of actinmicrofilaments in cells treated with spisulosine 285 and the lipidstructure of spisulosine 285 suggest that spisulosine compounds mayserve as an antagonist for the LPA receptor, preventing LPA interactingwith its receptor to activate Rho to produce the microfilaments.

The preferred compounds of this invention were initially isolated fromSpisula polynyma. Spisula polynyma is an edible clam, which is alsoknown as the Stimpson surf clam or the Atlantic surf clam. It belongs tothe subfamily Mactrinae, family Mactridae, superfamily Mactroidea, orderVeneroida, subclass Heterodonta, class Bivalvia, phylum Mollusca.Spisula polynyma was originally found off the coast of Japan, where itis called hokkigai and processed for sushi. It has now migrated throughthe Bering Strait, down past Greenland and Newfoundland, into theAtlantic ocean. The clam has a grey-white shell, 7–10 cm long. It ismainly off-white, except for the tongue which is purple in the livingclam, but turns bright red after cooking.

Thus, the present invention provides active extracts of the clam Spisulapolynyma. One embodiment of the present invention is directed to novelcompounds isolated from the clam Spisula polynyma, and the use of all ofthe cytotoxic compounds isolated therefrom as antitumor compounds.

To test for biological activity, one clam was homogenized in 3:1methanol/toluene. A solution of sodium chloride was added to this crudeextract, causing it to separate into a toluene and an aqueous layer. Thelatter was further extracted with toluene, dichloromethane, ethylacetate and 1-butanol. These extracts were all assayed against L1210cells, where significant cytotoxicity was observed for the initialcrude, toluene and dichloromethane extracts and less activity in theother three fractions.

L1210 Cytotoxicity of Crude Extracts of Spisula polynyma ^(a,b)Concentration (μg/ml) Extract 250  125  50 25 12.5 5 Crude  98*  98* 9225 0 0 Toluene 100* 100* 100* 25 13 13 CH₂Cl₂ 100* 100* 100* 91 20 13EtOAc  98*  98*  92* 0 0 0 1-Bu0H 83 33  0  0 0 0 Aqueous^(c) 94 75  0 0 0 0 Footnotes: ^(a)cytotoxity reported as % inhibition of growth;^(b)entries marked with * showed pointed cell activity; ^(c)the aqueousextract was assayed at 700, 350, 140 70, 35 and 14 mg/ml.

These extracts were also assayed against Herpes simplex virus Type 1(HSV-1) and CV-1 monkey kidney cells (at 100 mg/6.35-mm disk), but noactivity was observed. No antimicrobial activity was observed for theseextracts against Penicillium melinii (formerly P. atrovenetum) andMicrococcus luteus (formerly Sarcina lutea), both at 500mg/12.7-mm-disk. Later, other more purified extracts were assayedagainst Bacillus subtilis, Saccharomyces cerevisiae, and Escherichiacoli with no bioactivity observed.

Synthetic methods are also available for the preparation of spisulosinecompounds, particularly spisulosines 285 (1), 299 (2) and 313 (3).

The preferred synthetic route is based upon the previous addition oforganometallics to N,N-dibenzylamino aldehydes to yield β-amino alcoholswith high stereoselectivity. See, Andres et al., Org. Chem. 1996, 61,4210 and Reetz et al., Angew Chem. Int. Ed. Engl., 1987, 26, 1141. Thenon-chelation controlled addition of Grignard reagents or organolithiumcompounds produces the anti-diastereomer and the chelation controlledaddition of organozinc preferentially gives the syn-diastereomer.

Scheme I illustrates this preferred synthetic process for the formationof Compound 1:

As described in Scheme 1, the β-amino aldehyde 50 can be prepared fromL-alanine methyl ester by first dibenzylation of the amino group withbenzyl bromide and potassium carbonate followed by lithium aluminumhydride reduction to the N,N-dibenzylamino alcohol 40. The Swernoxidation of 40 gives 50 in high yield and can be used without furtherpurification to avoid decomposition. Addition of the Grignard reagent to50 gives the anti-diastereomer 60 with high selectivity. The compound,60, can be easily purified, for example by flash chromatography andHPLC. The deprotection of 60 by hydrogenolysis on Pearlman's catalystgives 1 in high yield and a good overall yield. Compounds 2 and 3 may beprepared simply by increasing the chain length of the Grignard reagent,and the remaining compounds of the present invention may also beprepared by appropriate choice of the Grignard reagent.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A, 1B, 1C, 1D, 1E and 1F are illustrations of the cellmorphologies observed in the L1210 assays of Spisula polynyma extracts.FIG. 1A represents a normal cell; FIG. 1B represents a typical pointedcell; FIG. 1C represents an atypical pointed cell; FIG. 1D represents acell with more than two points; FIG. 1E represents a bulged cell; andFIG. 1F represents a combined bulged and pointed cell.

FIG. 2 illustrates the scheme used to separate the compounds describedherein from extracts of the clam Spisula polynyma.

FIG. 3 is a microphotograph for the results in Example A.

FIG. 4 is a microphotograph for the results in Example B.

FIG. 5 is an electrophoretogram of Example C.

FIG. 6 is a microphotograph for the results in Example D.

DETAILED DESCRIPTION OF THE INVENTION

Referring to FIG. 1, in the L1210 assay some of the cells changed frombeing spherical (FIG. 1A) to ovoid with long points approximately 180°C. apart (FIG. 1B). Several other forms have also been observed inassays of these extracts, including cells with points not 180° C. apart(FIG. 1C), cells with more than two points (FIG. 1D), cells with a bulge(FIG. 1E) and cells with a bulge replacing one of the points (FIG. 1F).However, the form with two sharp, opposing points was by far thepredominant and characteristic one observed. This type of morphologicalchange had not previously been observed during the screening of over1000 marine extracts.

Isolation of Spisulosines 285, 299, and 313

For this invention, Spisula polynyma were collected, at a depth of −110feet, from a clam bed on the eastern edge of Stellwagon bank which islocated off the coast of New England, stretching from near Gloucester,Mass., north to Maine. They were shipped live by the New England ClamCorporation (formerly New Dawn Seafoods, Inc.) and then immediatelyfrozen.

A purification scheme similar to the extraction procedure describedabove for the original testing of the bioactivity was employed. First 35clams were thawed and the shells removed to give 1.9 kg (wet wt). Thesewere allowed to stand in 3:1 methanol/toluene and filtered after severalhours. This step was repeated followed by homogenization and extensiveextraction with this same solvent to give a crude extract. To this wasadded a 1 M sodium chloride solution which caused the extract toseparate into two layers. The lower aqueous layer was further extractedwith toluene and the toluene layers combined. The resulting aqueouslayer was then extracted with dichloromethane as shown in FIG. 2.

The toluene extract was partitioned between methanol and hexane. Thecytotoxicity and cellular alteration were observed almost exclusively inthe methanol fraction. The methanol extract thus obtained was applied toa silica flash column, eluting with a chloroform/methanol step gradient(100:0, 99:1, 95:5, 90:10, 85:15, 80:20, 70:30, 50:50, 0:100). The maincytotoxic and pointed-cell-forming activity eluted off the column verylate, although earlier fractions did show some cytotoxicity, but nopointed cells. This late eluting was further purified by flash silicachromatography, using 8:12:1:1 chloroform/1-butanol/acetic acid/water.Fractions were neutralized with sodium bicarbonate before removing thesolvent to prevent possible decomposition when they were concentrated inacid. This resulted in a series of three bioactive fractions.

It had been observed in earlier attempts at isolation that thebioactivity did not wash off of a cyano solid-phase extraction (SPE)column with methanol, but the cytotoxicity was found to elute with 3:1methanol/0.01 M ammonium formate (0.5 ml/min). This was confirmed bychromatographing a small amount of a bioactive fraction on a cyano HPLCcolumn with this same solvent system and then repeating the injectionunder the same conditions except replacing the ammonium formate solutionwith water. The chromatograms appeared identical except that a peakeluting at 15.6 min was only observed in the first.

The three bioactive fractions from the second silica column were eachfurther purified by cyano HPLC with the same conditions used above(except 1 ml/min) to give three series of bioactive fractions. Theammonium formate was removed by passing the sample through a C-18 SPEcolumn, washing first with water and then eluting with methanol. Themain cytotoxicity and morphology-changing activity of each series(fractions A, B, and C) was found in a peak comparable to that discussedabove. However the activity was spread throughout most of the fractions.Silica TLC (3:12:2:2 chloroform/1-butanol/acetic acid/water) indicatedthat fraction A (0.4 mg) contained one spot (R_(f) 0.47), which was pinkby ninhydrin. Fraction B (1.3 mg) showed this same spot as well as oneslightly lower (R_(f) 0.44, red by ninhydrin), while fraction C (0.2 mg)contained both of these and a third one (R_(f) 0.34, purple byninhydrin). All three showed good cytotoxicity and pointed-cell formingactivity, with A exhibiting slightly more activity than B andsignificantly more than C. This indicated that the uppermost TLC spotmust be from compound(s) which caused the morphological change in L1210cells. These fractions were not purified further, but analyzed asmixtures. Quantitative bioassay results are discussed below.

An attempt was made to determine if a particular organ of Spisulapolynyma contained most or all of the bioactivity. A live clam wasanesthetized with diethyl ether and then dissected into nine parts:foot, digestive system, gonads, siphon, gills, heart, mantle, adductormuscles, and the remainder of the visceral mass (with foot, digestivesystem and gonads removed). These were identified by comparison toillustrations of other clams. Each organ was homogenized in 3:1methanol/toluene and the resulting extract was then triturated withdichloromethane and methanol to remove salts. While all of the extractsshowed cytotoxicity (Table), only those from the gills and the gonadsexhibited strong morphology-changing activity. That from the digestivesystem and the remainder of the visceral mass also showed weakpointed-cell forming activity, possibly due to incomplete separationfrom the gonads. The lack of pointed-cell-forming activity in otherorgans may have resulted either from a lack of 1–3 or from a much lowerconcentration.

In another experiment, one foot that had been cooked for a brief periodwas extracted in an analogous manner. This also showed cytotoxicity, butno morphology-altering activity. However, when a larger sample of cookedmaterial was more extensively extracted, some pointed cells wereobserved in the L1210 assay. Silica TLC (3:12:2:2chloroform/1-butanol/acetic acid/water, 100 mg) of the extracts of thedigestive system and gonads showed a weak ninhydrin-positive spot atR_(f) 0.49.

250 μg/ml 125 μg/ml 50 μg/ml Organ % Inhibition % Pointed^(a) %Inhibition % Pointed^(a) % Inhibition % Pointed^(a) foot 100  0, ad^(b)100  0, ad 93  0, 0 digestive  96  0, 18  62  0, 0  0  0, 0 systemgonads nr^(c) nr  99 56, 100 90  32, 100 siphon 100 ad, ad  50  0, 0  0 0, 0 gills 100 ad, ad 100 50, ad 98 100, 93 heart 100 ad, ad nr  0, 038  0, 0 mantle 100  0, 0  99  0, 0 95  0, 0 adductor 100 added 100  0,0 95  0, 0 muscles visceral 100 10, ad 100  2, ad 94  0, 0 mass cooked100  0 100  0 97  0 foot^(d) cooked  91 21  25  0  0  0 foot^(e)footnotes: ^(a)Percentage of pointed cells was measured at 58 to 82 hafter the start of the assay. ^(b)ad = all dead. ^(c)nr = not read dueto precipitated material in the assay which obscured the cells.^(d)Percentage of pointed cells measured at 72 h after the start of theassay. ^(e)This sample was extracted in a similar manner to that used toobtain the crude extract of the isolation of fractions A–C. Percentageof pointed cells measured at 76 h after the start of the assay.

Several clues to the structure of the bioactive compounds could be foundin the isolation procedure. The TLC spot which correlated with theactivity visualized as pink or red by ninhydrin, suggesting that thecompounds contained primary amines. Also, they exhibited amphiphiliccharacter. They were originally extracted into toluene from aqueousmethanol, but they then partitioned into methanol versus hexane. Whilethey are soluble in nonpolar solvents, they require a very polar solvent(3:12:2:2 chloroform/1-butanol/acetic acid/water) to be eluted fromsilica.

Only fractions A and B were reasonably pure from inactive contaminantsas shown by TLC. Most of the structure determination studies werecarried out on fraction B because of its size relative to the others.FIGS. 3 and 4 show the ¹H NMR spectra of this fraction in CDCl₃ andCD₃OD, respectively. What was immediately obvious in these spectra was apeak corresponding to a long methylene chain (1.25 ppm) and severaloverlapping terminal methyl groups (0.87 ppm). Other peaks were not aswell defined. No peaks corresponding to aromatic protons were observed,but several peaks appeared in the alkene proton region. Several othersseemed to correspond to protons attached to heteroatom-substitutedcarbons. The major difference between the spectra in the two differentsolvents was that, in CD₃OD, a methyl doublet (1.21 ppm) downfield ofthe terminal methyl groups were clearly observed, while in CDCl₃ thisresonance appeared only as an upfield shoulder on the methylene chainpeak.

An authentic sample of D-trans-erythro-sphingosine (4) was obtained fromSigma for comparison with the isolated material. The ¹H NMR spectrumthereof was similar in many respects to that of fraction B. As expected,4 exhibited a large peak due to the long methylene chain (1.25 ppm), aterminal methyl group (0.87 ppm) and two vinyl protons (5.75 and 5.46ppm). Of particular note was the broadness of the resonancescorresponding to protons on the heteroatom-substituted carbons (4.40,3.66, 2.85 and 2.18 ppm). Also, on silica TLC (3:12:2:2chloroform/1-butanol/acetic acid/water), 4 had R_(f) 0.43 and appearedred by ninhydrin, like the lower spot in fraction B and the middle spotin C. Palmeta and Pro{hacek over (s)}tenik have reported that2-amino-3-octadecanol and 4 exhibited very similar R_(f) values (0.32and 0.29, respectively) when eluted on paper impregnated with silicicacid with the solvent system di-isobutyl ketone/acetic acid/water(40:25:5).

Fractions A-C were also studied by several mass spectrometric methods.The largest ion in all of the spectra was m/z 286. High resolutionmeasurement of this peak (m/z 286.3109) allowed the assignment of themolecular formula C₁₈H₄₀NO (Δ0.1 mmu) to spisulosine 285 (1). Thiscompound derived its name, in part, from its molecular weight. Thismolecular formula indicated that the molecule is totally saturated. Astrong peak corresponding to the loss of water from this M+H ion wasobserved at 268.3019 (Δ−1.5 mmu). Thus, 1 must contain a hydroxyl group.Ions corresponding to matrix adducts, of m/z 286 were observed at m/z438.3078 (C₂₂H₄₈NO₃S₂, Δ−0.2 mmu), 590, and 592.

One well-known primary metabolite that, like 1, consists of an 18 carbonchain substituted with hydroxyl and amine functionalities is sphingosine(4). This compound has one more oxygen and two less hydrogens than 1.The analogy appeared valid because high resolution measurement of m/z300 for the spisulosines indicated that it was a doublet correspondingto the M+H of a higher homologue (2) of m/z 286 (300.3270, C₁₉H₄₂NO,Δ−0.4 mmu), together with sphingosine (4) itself (300.2914, C₁₈H₃₈NO₂,Δ−1.1 mmu). This also helped to explain the presence of alkene protonsin the ¹H NMR spectrum.

Several other peaks were evident in all three spectra. The ion at m/z314 was also a doublet corresponding to C₂₀H₄₄NO (314.3439, Δ−1.6 mmu),which was the molecular ion of another homologue of 1, spisulosine 313(3), and C₁₉H₄₀NO₂ (314.3075, Δ−1.6 mmu) which was a homologue ofsphingosine (5). Compound 4 showed matrix adducts of the M+H ion at m/z452.2885 (C₂₂H₄₆NO₄S₂, Δ−1.7 mmu), 604.2831 (C₂₆H₅₄NO₆S₄, Δ0.3 mmu) and606.2995 (C₂₆H₅₆NO₆S₄, Δ3.6 mmu), 5 exhibited matrix adducts of the M+Hion at m/z 464.2888 (C₂₃H₄₆NO₄S₂, Δ−2.0 mmu) and 618.2940 (C₂₇H₅₆NO₆S₄,Δ5.1 mmu). It should be noted that, while m/z 300 and 314 were doubletsof nearly equal intensity in fraction B, only one peak was measurablefor the matrix adducts listed here from fraction B. This suggested thatthese two series of compounds, although very similar in generalstructure, behaved differently in FABMS. The spisulosine series(saturated) gave strong molecular ions and weaker matrix adducts, whilethe reverse was observed for the sphingosine series (unsaturated).

To better establish the structures identified by the data discussedabove, several derivatives were prepared. The most informative was thediacetyl derivative of spisulosine 285 (8). Because fraction B was thelargest, a portion of it was acetylated with acetic anhydride inpyridine. This mixture of acetyl derivatives will be referred to here asAcB. By silica TLC (3:12:2:2 chloroform/1-butanol/acetic acid/water),the reaction appeared quantitative, with a new spot appearing at R_(f)0.86. For comparison, the triacetyl derivative of authentic 4 (9) wasalso synthesized by the same method.

Two series of compounds related to the spisulosines have been previouslyisolated. Gulavita and Scheuer reported that a Xestospongia sp. spongefrom Papua-New Guinea contained two epimeric 14-carbon amino alcohols134 and 135. These were not isolated as the free amines, but rather themixture was acetylated to give both the mono- (136, 137) and diacetylcompounds (138, 139) which were then separated. Jimenez and Crews haveisolated several molecular ion of the underivatized 1 at m/z 286. ThisM+H ion (m/z 370) fragmented to give m/z 310 and 268, presumably bylosing acetic acid and then the second acetyl group, respectively. Thecomparable ions for the other spisulosines were small, but present: m/z384, 324 and 282 for the diacetyl derivative of 2 (144), and m/z 398,338 and 296 for the diacetyl derivative of 3 (145). The ions from thesphingosine in the sample were too small to state definitively that theywere present. This again showed that the two series of compounds hadvery different ionization potentials. The CIMS spectrum showed strongm/z 370 and 310 ions, but here the m/z 268 ion was very weak. The higherhomologues were again seen at m/z 384 and 324 for 144, and m/z 398 and338 for 145. Weak ions at m/z 426 and 366 were indicative of 133.

Synthesis of Spisulosine 285

To confirm the structure and determine the stereochemistry ofspisulosine 285 the compound was synthesized. None of the isomers of2-amino-3-octadecanol were previously known as natural products, butboth the 2S, 3S and 2S, 3R isomers have previously been synthesized. Thehigher homologues are novel compounds.

A modified version of the synthesis of Pro{hacek over (s)}tenik andAlaupovic (Scheme IX) was used to obtain the authentic material forcomparison.

First, dibenzyl malonate (147) was alkylated with tetradecyl bromide(148). The resulting dibenzyl tetradecylamalonate (149) was thencondensed with N-phthaloyl-L-alanyl chloride (150) to give2-phthalimido-3-octadecanone (151) after removal of the benzyl groupsand decarboxylation. This ketone was treated with excess sodiumborohydride, which resulted in the reduction of one of the phthalimidocarbonyls in addition to the ketone, producing both 152, which had onephthalimido carbonyl reduced to the carbinolamine, and 153, which wasfurther reduced. These two products could be readily separated from eachother by silica flash chromatography.

The reduction of 151 to 152 produced a mixture of four diastereomersbecause of the formation of two new chiral centers. At this point, thediastereomers were separated by cyano HPLC. The protecting group wasthen removed from each by further reduction with sodium borohydridefollowed by acetic acid. As one stereocentre was removed with theprotecting group, this resulted in the production of two diastereomers.Since this synthesis started with L-alanine, the two products were(2S,3S)-2-amino-3-octadecanol (154) and (2S,3R)-2-amino-3-octadecanol(155).

Biological Activity

While the spisulosines were quite simple compounds, as illustrated inFIGS. 1A–1F, they exhibited a very unusual type of bioactivity. Asdiscussed above, the spisulosines caused a distinct morphological changein L1210 leukemia cells, in addition to cytotoxicity. This bioactivity,which was recorded as the percentage of living cells in which alteredmorphology was observed, could be observed sometimes as early as 13 hafter the start of the assay and reached a maximum at 50–60 h, afterwhich it decreased. Generally 60 cells were observed to determine thisnumber, except in assays in which less than this number of cellsremained alive. The morphological effect was usually measured 30–35 hafter the start of the assay and again about 24 h later, while thecytotoxicity was determined when the number of cells in the controlsreached approximately 8000, usually in 3 days after the assay was begun.It should be noted that the pointed cells were live cells and that theywere counted as such for the cytotoxicity reading. Also, assays in which100% cytotoxicity was recorded may still have contained live cells(<0.5%) which may or may not have been pointed. Allmorphologically-changed cells were counted in the pointed cellpercentage.

This change in morphology was always observed in fractions with fairlyhigh cytotoxicity. Generally, no significant number of pointed cellswere observed in assays with less than 70% growth inhibition. However,assays in which the cytotoxicity approached 100% often had lowerpercentages of cells with altered morphology than those with 90–98%growth inhibition. This suggested that the altered cells might be moreeasily killed. It is unknown whether the cytotoxicity and the morphologychange resulted from the same mechanism of action. In one instance,pointed cells from an assay were recultured and found to revert to thenormal state. This suggested that the effect was reversible after thecompound had been metabolized. Acetylation drastically reduces thebioactivity.

To determine if the change in morphology of L1210 cells was caused bysphingosine (4) or related compounds, several authentic compounds wereobtained and assayed against L1210 cells. Both sphingosine andstearylamine (131) exhibited moderate cytotoxicity, but no morphologicaleffect. Sphingomyelins are well-known derivatives of 4 in which aphosphoryl choline unit has been added to the primary alcohol and theamine is acylated by a fatty acid. A mixture of sphingomyelins isolatedfrom bovine brain (Sigma), which consisted mainly of stearoyl andnervonoyl sphingomyelins (161, 162), showed minimal cytotoxicity and nopointed cells. The cytotoxicity of the phosphoryl choline derivative of4 (163, Sigma) may be, at least, partially due to hydrolysis of 163 to4.

Cytotoxicity of Model Compounds Concentration % % Pointed Compound(μg/ml) Inhibition cells 128 5 100 0 2.5 100 0 1 75 0 0.5 31 0 0.25 13 00.1 0 0 161 + 162 50 7 0 25 0 0 10 0 0 131 5 99 0 2.5 96 0 1 19 0 0.5 00 0.25 0 0 0.1 0 0 163 50 88 0 25 50 0 10 38 0

Sphingosine and other long-chain amines, including stearylamine, areknown to be cytotoxic. This bioactivity, as measured against Chinesehamster ovary (CHO) cells, has been shown to be maximal for 18-carbonhomologues. All four stereoisomers of sphingosine were found to bealmost equally active. Reduction of the double bond of 4 to producedihydrosphingosine (164) did not affect the cytotoxicity. Addition of anN-methyl group to 164 also caused no significant change in thebioactivity, while acylation of the amine caused a large decrease in thecytotoxicity.

No cytotoxicity was reported for the related compounds (134, 135,140–142) which have been isolated from other marine sources, however,they may not have been tested in this type of assay. A mixture of 134and 135 was active against C. albicans (8-mm zone of inhibition for 19mg of a mixture of the two). Xestaminol A was reported to exhibit weakactivity against several Gram-positive and Gram-negative bacteria andfungi. It also showed antihelminthic activity against Nippostrongylusbrasiliensis. Both 140 and 142 showed some activity against reversetranscriptase.

The activity of fractions A–C, the acetyl derivative of fraction B andcompounds 154 and 155 is summarized in the table. The assay resultsclearly confirmed the NMR analysis assigning 155, not 154, as the sameas 125. Also, acetylation drastically reduces the bioactivity.

TABLE IX Bioactivity of Fractions A–C, AcB, and 154 and 155Concentration% % Pointed Cells^(a/b) Sample (μg/ml) Inhibitiion^(c) Time1st 2nd Fraction A 2.5 100 35, 59 ad ad 1.25 100 25  ad 0.5 90 42  450.25 85 45  55 0.125 75 8 35 0.05 19 0  0 Fraction B 2.5 100 35, 59 0  71.25 93 3 21 0.5 90 2 43 0.25 80 7 37 0.125 75 5 21 0.05 7 0  0 FractionC 2.5 90 55 0 1.25 88 0 0.5 63 0 AcB 10 31 27 0 5 38 0 2 13 0 1 0 0 0.50 0 0.2 0 0 154 5 100 27 0 2.5 100 0 1 63 0 0.5 0 0 0.25 0 0 0.1 0 0 1555 100 27 ad 2.5 100 22  1 100 64  0.5 99 56  0.25 96 40  0.1 63 33 footnote ^(a)Unless otherwise indicated, the percentage of pointed cellswas read twice. The number of hours after the start of the assay atwhich these measurements were made is indicated in the time column.^(b)ad = all dead. ^(c)The percentage of growth inhibition which wasrecorded as the percentage of live cells in the treated wells comparedto that in control wells.Possible Mode of Action

The bioactivity of the spisulosines may be due to their similarity tosphingosine. In the nomenclature of sphingolipids, spisulosine 285 wouldbe considered 1-deoxysphinganine. The spisulosines may compete withsphingosine for binding sites or be incorporated into sphingolipids suchas sphingomyelins, ceramides or gangliosides. In either case. thespisulosines could disrupt the cellular functions controlled by thesecompounds. Sphingosine and its derivatives are involved in theregulation of cell growth and differentiation. Sphingosine is a potentinhibitor of protein kinase C, competing with diacylglycerol for thebinding site, which may explain its cytotoxicity. Structure-activitystudies have shown that this inhibition requires a positively chargedamine and thus N-acyl derivatives were inactive. If the spisulosines actby competing with sphingosine, this would explain the relative lack ofactivity of the acetylated compounds (AcB). There is growing evidencethat sphingosine may act as a second messenger by regulating proteinkinase C activity. It has also been shown to inhibit the differentiationof HL-60 cells treated with phorbol 12-myristate-13-acetate, a knownprotein kinase C activator. The spisulosines should be tested forinhibition of protein kinase C. It is unknown whether inhibition of thisenzyme could cause the morphological effects observed for thespisulosines, but protein kinase C is involved in the control of cellgrowth and differentiation.

Experimental

NMR spectra were obtained on General Electric GN 500 and QE 300 andVarian U400 spectrometers. Samples for NMR analysis were dissolved inCDCl₃ or CD₃OD. Chemical shifts (δ) are reported in ppm downfield oftetramethylsilane (TMS) and referenced to the residual solvent peak orTMS. Low and high resolution FABMS spectra were recorded on either a VGZAB-SE or a VG 70-SE4F spectrometer, using a 3:1 mixture ofdithiothreitol-dithioerythritol (magic bullet) as the matrix. FABMS/MSspectra were recorded on a VG 70-SE4F with the same matrix, using heliumas the collision gas. Cl mass spectra were recorded on a VG VSEspectrometer, operating in the alternating Cl/El mode with methane asthe reagent gas. IR spectra were obtained on an IBM IR/32 FTIRspectrometer. Optical rotations were measured on a JASCO DIP-370 digitalpolarimeter.

Chromatography

HPLC was carried out using an Alltech Econosphere cyano column (4.6×250mm, 5 gm particle size). The HPLC system used consisted of a BeckmanModel 114M pump, a Rheodyne 71 injector and either an Isco V⁴ or Beckman165 variable wavelength detector or a Waters 990 photodiode arraydetector.

Analytical thin layer chromatography (TLC) was performed on a pre-coatedsilica gel (Merck 60 F-254) and cyano bonded-phase (EM Science CNF_(254S) HPTLC) plates. Spots were visualized by UV (254 nm), ninhydrin(5% in ethanol), phosphomolybdic acid (5% in ethanol) and/or iodine.Silica column chromatography was carried out on either 50–200 mm or40–63 mm silica gel (Merck). Other column chromatography usedChromatorex ODS (Fuji-Division 100–200 mesh) and Sephadex LH-20(Pharmacia). High speed countercurrent chromatography (HSCCC) wasperformed on an Ito multi-layer coil separator-extractor (P.C., Inc.)with a #10 coil and a Milton-Roy mini-Pump. Solid phase extraction (SPE)was carried out on normal phase (silica, Alltech Maxi-Clean),reversed-phase (C-18, Waters Sep-Pak), and bonded-phase (CN, FisherPrepSep) columns.

Biological Assays

Cytotoxicity assays against L1210 murine lymphocytic leukemia cells wereperformed by dissolving the samples in methanol and/or hexane wereapplied to the dry assay wells and the solvent was allowed to evaporate.Cells (1000) were added in minimum essential medium (MEM, 1 ml) andincubated 37° C. Inhibition of growth was recorded as the estimatedpercentage of living cells in sample wells versus that in control wells.This was measured when the control wells reached 8000 cells, generallythree days after the start of the assay.

Morphologically-changed cells (FIGS. 1A–1F) were counted as living cellswhen determining the percent inhibition of growth. Morphological changeswere assessed throughout the assay period. The percentage of pointedcells was determined by counting the number of altered cells inapproximately 60 living cells. This percentage varied with the length oftime the assay had been running. It generally reached its maximum about50 hours after the start of the assay, but pointed cells could beobserved as early as 13 hours after the start of the assay and couldusually still be seen when the percent growth inhibition was measured.The percentages of pointed cells were often counted both after about 35and after 55 hours. The time that this measurement was made is indicatedwith the data.

Antimicrobial assays were performed using the filter disk diffusionmethod. Paper disks (6.35 or 12.7 mm, Schleicher & Schuell) wereimpregnated with samples (50–500 μg) in solution and allowed to dry.These disks were then placed on agar seeded with either Bacillussubtilis, Penicillium melinii (formerly P. atrovenetum), Micrococcusluteus (formerly Sarcina lutea), Escherichia coli or Saccharomycescerevisiae. These plates were incubated for 12–24 h (32–35° C., exceptP. melinii, 25–27° C.).

Extraction of Spisula polynyma for Initial Biological Testing

One clam (Spisula polynyma) was thawed and the shell removed (35.32 g,wet wt). This was placed in a blender with 350 ml of 3:1methanol/toluene and homogenized. The yellow-brown extract was filteredand added to a 1 M sodium chloride solution (100 ml). The upper toluenelayer was removed and the aqueous layer extracted with toluene (75 ml).The two toluene layers were combined and the solvent was removed to givea brown oily residue (333.9 mg). The aqueous layer was further extractedwith dichloromethane (2.times.75 ml), which gave a yellow-brown residue(18.6 mg) after removal of the solvent. The aqueous layer was thenextracted with ethyl acetate (75 ml). The lower phase was the organiclayer due to the presence of some dichloromethane which had remained inthe aqueous phase after the last step. The upper layer was furtherextracted with the ethyl acetate (245 ml), the upper organic layerback-extracted with water (100 ml), and the two ethyl acetate extractswere combined to give a yellow residue (36.8 mg) after removal of thesolvent. The combined aqueous layers were concentrated by one-half andextracted twice with 1-butanol (150 ml, 75 ml). The combined butanollayers were back-extracted with water (75 ml), resulting in a yellowresidue (132.8 mg) after removal of the butanol. The combined aqueouslayers were concentrated to give an oily light yellow residue (946.1mg). Each extract was triturated with dichloromethane and methanol toremove salts to give the toluene (302.2 mg), dichloromethane (18.6 mg),ethyl acetate (36.7 mg), butanol (120.9 mg) and aqueous (590.4 mg)extracts which were assayed.

Fractions A, B, and C

Thirty-five clams were thawed and the shells removed to give a sample ofSpisula polynyma (1.9 kg) which was soaked in methanol/toluene (3:1,2×1.51). The solids were then ground in the same solvent (6×1.51) andthe resulting extracts filtered. A 1 M solution of sodium chloride (31)was added to this crude extract (121) and the resulting upper toluenelayer removed. The aqueous layer was further extracted with toluene(2×2.51), followed by dichloromethane (4×2.51) as shown in FIG. 1.

After removal of the solvent, the toluene extract (21.55 g) waspartitioned between methanol and hexane (1.51 each). The methanol layerwas further extracted with hexane (4×11). The combined hexane layerswere concentrated to about 1.81 and both extracts chilled (−10° C.). Thetwo layers which resulted in each case were separated. The combinedhexane layers were then extracted with methanol (0.51). This processresulted in a hexane and three methanol extracts of which the firstmethanol extract (6.8 g) contained the most bioactivity.

This bioactive methanol fraction was separated by flash silicachromatography employing a chloroform/methanol step gradient (100:0,99:1, 95:5, 90:10, 85:15, 80:20, 70:30, 50:50, 0:100) to give 12fractions. While the third, fourth, seventh and eighth fractionspossessed some cytotoxicity, they showed no pointed-cell formingactivity. This activity was found in the last two fractions along withmost of the cytotoxicity.

These two fractions were combined (370 mg) and further purified byanother flash silica column, using chloroform/1-butanol/aceticacid/water (8:12:1:1). To remove the acetic acid, each of the 12fractions thus obtained was neutralized by (a) adding chloroform(one-quarter volume), (b) washing with 5% sodium bicarbonate until thepH of the aqueous layer was above 7 (2–3×half volume), and then (c)washing the organic layer with water (half volume). The third, andfourth and fifth fractions possessed all of the pointed cell-formingactivity and essentially all of the cytotoxicity. Each of thesefractions was separately purified by HPLC on a cyano column with 3:1methanol/0.01 M ammonium formate (1 ml/min). Six fractions, of which themost bioactive was the fifth, were collected from each silica fraction.The ammonium formate was removed from each fraction by adding water (2–8ml), applying the sample to an SPE column (C-18), washing with water(5–10 ml) and then eluting with methanol (5 ml). This resulted infractions A (0.4 mg, 2×10⁻⁵% yield), B (1.3 mg, 7×10⁻⁵% yield) and C(0.2 mg, 1×10⁻⁵% yield), from the third, fourth and fifth silicafractions, respectively, which all eluted at t_(r) 7.9 min.

Fraction A

White solid; silica TLC (3:12:2:2 CHCl₃/1-BuOH/AcOH/H₂O)R_(f) 0.47(ninhydrin-positive, pink); IR (NaCl) 2922, 2853, 1734, 1593, 1462,1377, 1061 cm⁻¹; ¹H NMR (CDCl₃) d 5.38, 5.15, 3.82, 3.67, 3.44, 3.24,2.31, 2.03, 1.67, 1.60, 1.55, 1.25, 1.10, 0.86; FABMS m/z 606, 604, 592,590, 466, 452, 438, 314, 300, 286, 268; CIMS m/z 354, 340, 338, 328,326, 324, 314, 312, 310, 300, 298, 296, 286, 284, 268, 266, 149, 139,137, 1, 123, 111, 109, 97, 95, 85, 83, 71, 69, 59, 57, 55. Anal. Calcd.For C₈H₄₀NO: 286.3110 (M+H). Found: 286.3115 (HRFABMS).

Fraction B

White solid; silica TLC (3:12:2:2 CHCl₃/1-BuOH/AcOH/H₂O) R_(f) 0.47(ninhydrin-positive, pink), 0.44 (ninhydrin-positive, red); IR (NaCl)3273, 2953, 2918, 2851, 1639, 1591, 1510, 1466, 1379, 1344, 1059, 970cm⁻¹; ¹H NMR (CDCl₃) δ 5.98, 5.78, 5.55, 5.44, 5.32, 4.43, 3.78, 3.65,3.24, 2.15, 2.08, 2.00, 1.95, 1.70, 1.44, 1.25, 1.19, 0.87; FABMS m/z6.18.2940, 616, 606.2955, 604.2831, 592, 590, 480, 466, 464.2888,452.2885, 438, 314.3439, 314.3075, 300.3273. 300.2914, 286, 268; CIMSm/z 354, 352, 342, 340, 338, 328, 326, 324, 314, 312, 310, 300, 298,296, 286, 284, 282, 280, 268, 266, 219, 193, 179, 165, 149, 137, 123,111, 109, 97, 95, 85, 83, 71, 69, 59, 57, 55.

Fraction C

White solid; silica TLC (3:12:2:2 CHCl₃/1-BuOH/AcOH/H₂O) R_(f) 0.47(ninhydrin-positive, pink), 0.44 (ninhydrin-positive, red), 0.34(ninhydrin-positive, purple); IR (NaCl) 2924, 2853, 1593, 1456, 1352,1063, 972 cm⁻¹; FABMS m/z 620, 618, 616, 606, 604, 602, 466, 464, 452,438, 314, 300, 298.2741, 296, 286, 280, 268; CIMS m/z 354, 352, 340,338, 336, 328, 326, 324, 322, 314, 312, 310, 308, 300, 298, 296, 294,292, 286, 284, 282, 280, 278, 268, 179, 165, 149, 137, 135, 1, 123, 121,111, 109, 97, 95, 85, 83, 81, 71, 69, 60, 59, 57, 55.

Initial Partitioning

Twenty-two S. polynyma claims were thawed and the shells removed to give1.3 kg of the organism (wet wt). This was placed in Waring blender with3:1 methanol/toluene (1.5 1) and ground into a thick slurry which wasfiltered through a layer of celite. The solid residue was furtherextracted (4×1.5 1) and filtered in a similar manner. The remainingsolids were then placed in 5:1 methanol/toluene (750 ml) and allowed tosoak for 36 h, before filtering. To the combined filtrates (7.8 1) wasadded 1 M sodium chloride (2 1). After removal of the upper toluenelayer, the aqueous phase was extracted with toluene (2×1.5 1) anddichloromethane (3×1.5 1). The remaining aqueous phase was concentratedby one-half and extracted with ethyl acetate (2×1 1). The resultingaqueous layer was diluted with water (2 1) and extracted twice with1-butanol (1.5 1, 1 1). Removal of the solvents and trituration withdichloromethane and methanol resulted in the toluene (14.1 g),dichloromethane (0.75 g), ethyl acetate (1.3 g), 1-butanol (0.2 g) andaqueous (1.9 g) extracts which were assayed.

The toluene extract was partitioned between hexane and methanol (750 mleach). The resulting methanol layer was further extracted with hexane(2×750 ml, 2×500 ml). The hexane layers were combined and concentratedto about 3 1 and then both extracts were chilled (−10° C.) which causedeach to separate into two layers. The combined methanol layers wereconcentrated in vacuo to give a brown residue (methanol extract 1, 536g). The hexane layers were further concentrated to about 1 1 andback-extracted with methanol (500 ml). The solvent was removed from eachof these to give the methanol extract 2 (4.26 g) and the hexane extract(4.52 g).

Fraction D

A portion of the first methanol extract (594 mg) was separated by HSCCC,using hexane/ethyl acetate/methanol/water (4:7:4:3, MP=UP) at 4 ml/min.This gave 12 fractions of which the third, fourth and fifth containedmost of the bioactivity. These three fractions were combined (158 mg)and chromatographed on Sephadex LH-20, eluting with methanol. Thisresulted in eight fractions of which the fourth (8.4 mg) possessed themajority of the biological activity. This bioactive fraction was furtherpurified by HPLC on a cyano column with 3:1 methanol/0.01 M ammoniumformate (0.5 ml/min). Eight fractions were collected and the ammoniumformate was removed from each by adding water (2–8 ml), applying thesample to an SPE column (C-18), washing with water (5–10 ml) and theneluting with methanol (5 ml). The seventh fraction (t_(r) 15.8 min,white amorphous solid, 0.3 mg, 2×10⁻⁴% yield) proved to contain thebioactive compounds and is referred to here as fraction D. Silica TLC(1-BuOH/AcOH/H₂O, 4:1:5, upper layer) showed four spots byphosphomolybdic acid visualization: R_(f) 0.53 (major), 0.35 (major),0.31 (minor), and 0.19 (minor). The inactive sixth fraction showed allthe same spots except R_(f) 0.53. The FABMS spectrum of fraction Dshowed intense peaks at m/z 286.3019, 300.3270 and 268.3019, and weakerpeaks at m/z 314, 438, 452, 464, 590, 592, 669, 797, 809 and 825. Thelast three ions listed were also observed in most of the other HPLCfractions and appeared to correspond to the TLC spot at R_(f) 0.35.

Anal. Calcd. for C₁₈H₄₀NO: 286.3110 (M+H). Found: 286.3109 (HRFABMS).

Fraction E

A second portion of the first methanol extract described above (633 mg)was subjected to HSCCC. The solvent system employed washexane/methanol/water (5:4:1, UP=MP, 5 ml/min), which gave poorstationary phase retention. This resulted in 10 fractions with thebioactivity spread throughout most of them. The first three fractions(310 mg) were combined and further purified by HSCCC using hexane/ethylacetate/methanol/water (4:7:4:3, LP=MP, 2 ml/min) to give 12 fractions.The second to fifth fractions (85 mg), containing the majority of thebioactivity, were chromatographed on a C-18 flash column, eluting with amethanol/water/chloroform step gradient (90:10:0, 95:5:0, 100:0:0,95:0:5, 90:0:10, 50:0:50). This gave 10 fractions which were allbioactive.

The fourth to sixth fractions from the first HSCCC run were combinedwith a side fraction from the Sephadex LH-20 column discussed underfraction D (270 mg). This material was subjected to HSCCC, using thesame conditions as the second run just described except that the flowrate was 3 ml/min. This resulted in nine fractions of which the secondand third contained most of the cytotoxicity and cell-altering activity.These two fractions were combined (42 mg) and separated on a flash C-18column, using a methanol/water step gradient (80:20, 90:10, 95:5,100:0). This resulted in 12 fractions of which the eighth to eleventhshowed morphology-altering activity and cytotoxicity. All but the firstand fifth fractions from the first C-18 column were combined with theeighth to eleventh fractions from the second (50.4 mg) and separated bypreparative silica TLC with chloroform/1-butanol/acetic acid/water(3:12:2:2). The plate was divided into eight fractions, which werescraped off and eluted with methanol. The residue from each fractionafter removal of the solvent was triturated with dichloromethane andfiltered. The fraction second from the top of the plate (R_(f)0.80–0.42) contained the bioactive material and is referred to asfraction E (5.7 mg). Analytical silica TLC of fraction E, eluting withthe same solvent system, showed a single spot by ninhydrin visualization(R_(f) 0.44), but phosphomolybdic acid spray regent showed othermaterial which streaked throughout the middle third of the plate. TheFABMS spectrum of fraction B showed m/z 286 as the major peak, withlesser peaks at m/z 268, 300, 438, 452, and 592.

Fraction F

A third portion of the first methanol extract (468 mg) was separated byflash silica chromatography, using the solvent systemchloroform/1-butanol/acetic acid/water (8:12:1:1). To remove the aceticacid, each of the 10 fractions thus obtained was neutralized by (a)adding water (half the volume of the fraction) and separating the twophases, (b) extracting the aqueous layer with chloroform (halfvolume×2), (c) washing the combined organic layers with 5% sodiumbicarbonate until the pH of the aqueous layer was above 7 (2 to 3×halfvolume), and then (d) washing the organic layer with water (halfvolume). The third fraction (24 mg), which possessed the majority of thebioactivity, was chromatographed on Sephadex LH-20, eluting withmethanol, to give eight fractions. The sixth fraction (2.3 mg) wasseparated by repeated HPLC, using the same conditions as for theseparation of fraction A-C. The ammonium formate was removed as forfraction A-C. The fraction eluting at t_(r) 8.1 min was the mostbiologically active and is referred to as a fraction F. It was so smallthat an accurate weight could not be obtained, but probably was 100–200μg (approximately 1 to 2×10⁻⁴% yield). The fractions eluting later thanthis one also showed both cytotoxic and pointed cell-forming activity,although less potent. This suggested that either the bioactivecompound(s) did not elute as a well-defined peak or that differenthomologues eluted at different times, but were not well separated.Silica TLC (3:12:2:2 CHCl₃/1-BuOH/AcOH/H₂O) showed oneninhydrin-positive spot at R_(f) 0.44. The later eluting fractions alsoshowed this same spot, but less intense. The FABMS spectrum of fractionF shows (in decreasing order of intensity) m/z 286, 268, 300, 314, 344,438, 452, 592, 669.

Dissection

A live clam was placed in a container with about 10 ml of diethyl etherand chilled (4° C.) for 20 h. It was dissected into nine organs: foot,digestive system (including the stomach, intestines and crystallinestyle sac), gonads, siphon, gills, heart, mantle, adductor muscles, andthe remainder of the visceral mass. Each organ was first soaked inmethanol/toluene (3:1, 10 ml/g sample) and then homogenized in a Virtisblender. The extracts were filtered and the solvent was removed. Theresidue was triturated with dichloromethane and methanol to give 155 mg(foot), 60 mg (digestive system), 147 mg (gonads), 101 mg (siphon) 65 mg(gills), 2.5 mg (heart), 168 mg (mantle), 101 mg (adductor muscles) and252 mg (visceral mass).

In a separate experiment, one foot that had been cooked was extracted inan analogous fashion (189 mg). A larger sample of cooked clams (483 g)was more extensively extracted by first soaking in 3:1 methanol/toluene(3×500 ml) and then homogenizing the sample in the same solvents (5×500ml). A small sample of the combined extracts was evaporated andredissolved in methanol for assaying.

General Procedures

Optical rotations were measured on a Jasco DIP-370 digital polarimeter,with a 3.5×50 mm 1 ml cell. Melting points were taken on a Thomas Hoovercapillary melting point apparatus. ¹H and ¹³C NMR were recorded on aVarian Unity-400 or Unity-500 spectrophotometer. Chemical shifts arereported in ppm relative to the solvent (7.26, CDCl₃ and 3.30, CD₃OD).High resolution (HRFAB) and fast atom bombardment (FAB) mass spectrawere recorded on a VG ZAB-SE or a 70 SE4F mass spectrometer. TLC wasdone on Merck Silica Gel 60 Thin-Layer Plates. Chromatographicseparations were done by flash chromatography using 230–400 mesh Mercksilica gel. All moisture sensitive reactions were run in oven-driedglassware under an atmosphere of N₂. Solvents were distilled prior touse: THF from benzophenone ketyl, CH₂Cl₂ from CaH₂ other solvents usedwere reagent grade.

(S)-2-(N,N-Dibenzylamino)propionic acid methyl ester (30)

To a 300 ml round bottom was added 20 (10.0 g, 71.6 mmol), benzylbromide (25.73 g, 150.4 mmol), K₂CO₃ (9.90 g, 71.6 mmol) and CH₃CN (172ml). The mixture was stirred at 60° C. until the reaction was completeby TLC. The reaction was cooler to room temperature and the solid wasseparated by filtration. The filtrate was concentrated in vacuo to givean oil which was purified by flash chromatography on silica gel (9:1hexane/EtOAc) to give a colorless oil: [α]² _(D)=113.6 (c 1.2, CHCl₃);¹H NMR (400 MHz, CDCl₃) δ 1.35 (d, 3H, J=7.1 Hz), 3.53 (q, 1H, J=7.0Hz), 3.65 (d, 2H, J=1.38 Hz), 3.75 (s, 3H), 3.85 (d, 2H, J=13.8 Hz),7.22–7.42 (m, 10H); ¹³C NMR (100 MHz) δ 14.9, 51.1, 54.3, 56.0, 2.8,4.1, 4.5, 139.1, 175.1; FABMS m/z 284.1 (M+H), 282.1 (M−H), 224.2(M-COOCH₃); HRFABMS calcd for C₁₈H₂₂NO₂M_(r) 284.165.1 (M+H), foundM_(r) 284.1650.

(S)-2-(N,N-Dibenzylamino)-1-propanol (40)

To a suspension of LiAlH₄ (550 mg, 14.5 mmol) in THF (20 ml) a solutionof 30 (910 mg, 3.21 mmol) in THF (2 ml) was added dropwise. The solutionwas stirred for 15 minutes and then heated to 65° C. for 3 hours. Thereaction was cooled to 0° C. and quenched with 0.1 N HCl. The reactionwas filtered through Celite and the Celite washed with THF (2×15 ml) andthe solvent removed in vacuo. Flash chromatography on silica gel (4:1hexane/EtOAc, R_(f)=0.30) gave 750 mg (92% yield) of a colorless solid:mp 40–41° C. (from hexane) Literature mp 40–41° C. (from hexane) See,Stanfield et al., J Org. Chem. 1981, 49, 4799–4800; [α]²⁵ _(D)=+86.6 (c1, CHCl₃) Literature [α]²⁵ _(D)=+88.2 (c 1, CHCl3); ¹H NMR (500 MHzCDCl₃) δ 0.98 (m, 3H), 2.98 (m, 1H), 3.13 (m, 1H), 3.35 (m, 3H), 3.45(m, 1H), 3.81 (m, 2H), 7.19–7.41 (m. 10H); ¹³C NMR (1 MHz) δ 8.6, 52.9,54.1, 62.7, 3.2, 4.5, 5.0, 5.3; FABMS m/z 256.2 (M+H), 224.2 (M-CH₂OH);HRFABMS calcd for C₁₇H₂₂NO M_(r) 256.1701 (M+H), found M_(r) 256.1702.

(S)-2-(N,N-Dibenzylamino)propionaldehyde (50)

Dry DMSO (0.53 ml, 7.43 mmol) was added to a stirred solution of oxalylchloride (0.31 ml, 3.6 mmol) in CH₂Cl₂ (7.5 ml) at −78° C. The solutionwas allowed to stir 15 minutes followed by the addition of 40 (740 mg,2.90 mmol) in CH₂Cl₂ (7.5 ml). After 30 minutes, Et₃N (1.0 ml, 7.2 mmol)was added and allowed to warm to room temperature. The solution wasextracted with saturated. NaHCO₃ (20 ml) and the aqueous layer wasextracted with CH₂Cl₂ (2×15 ml). The organic layer was washed withsaturated. NaCl solution, dried with MgSO₄ an concentrated in vacuo atroom temperature to give 720 mg (98% yield) of a yellow oil which becamea solid when cooled to −20° C. The aldehyde was used without furtherpurification: mp 52–54° C., Literature mp 55.5° C. See, Dix et al., ArchPharm (Weinheim) 1995, 328, 203–205.; [α]²⁶ _(D)=−36.0 (c 1, CHCl₃)Literature [α]²⁰ _(D)=−35.1 (c 1, EtOAc); ¹H NMR (400 MHz, CDCl₃) d 1.19(d, 2H, J=7.0 Hz), 3.34 (q, 1H, J=7.0 Hz), 3.58 (δ, 2H, J=13.7 Hz), 3.74(d, 2H, J=13.7 Hz), 7.26 (m, 2H), 7.33 (m, H), 7.42 (m, 4H), 9.74 (s,1H); ¹³C NMR (100 MHz) δ 6.7, 54.9, 62.8, 3.3, 4.4, 4.8, 139.1, 204.6;FABMS m/z 408.2 (M+MB), 254.2 (M+H), 22.2 (M-CHO); HRFABMS calcd forC₁₇H₂₀NO M_(r) 254.1545 (M+H), found M_(r) 254.1545.

(2S,3R)-2-(N,N-Dibenzylamino)-3-octadecanol (60)

Mg ribbon (237 mg, 9.75 mmol), dibromoethane (16 μL, 0.189 mmol) in THF(160 μL) were added to a two neck flask fitted with a reflux condenser.A ½ ml of a 1-bromopentadecane solution (970 mg, 3.33 mmol, 3.25 ml THF)was added. After the reaction had started the remainder was addeddropwise. To the grayish solution, 50 (105 mg, 0.413 mmol) in THF (0.5ml) was added dropwise. The reaction was allowed to stir overnightfollowed by the addition of H₂O (5 ml) and 0.1 N HCl until the solutionbecame clear. The mixture was extracted with EtOAc (3×10 ml). Theorganic layer was washed with 5% NaHCO₃ then saturated. NaCl solutionsand dried with MgSO₄. The solvent was removed in vacuo to give anoil-solid mixture (750 mg). The crude material was purified by flashchromatography on silica (8:1 hexane/EtOAc, R_(f)=0.34) to give 120 mgof a solid. This solid was further purified by HPLC on silica (93:7hexane/EtOAc) to give a colorless waxy solid (94.3 mg, 49% yield): [α]²⁵_(D)=+16.3 (c 1, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ 0.88 (t, 3H, J=7.0Hz), 1.10 (d, 3H, J=6.7 Hz), 1.16–1.41 (bm, 26H), 1.56 (m, 1H), 1.69 (m,1H), 1.79 (m, 1H), 2.72 (quin, 1H, J=6.7 Hz), 3.47 (d, 2H, J=13.8 Hz),3.60 (m, 1H), 3.76 (d, 2H, J=13.8 Hz), 7.22 (m, 2H), 7.30 (m, 4H), 7.34(m, 4H); ¹³C NMR (1 MHz) δ 8.67, 14.11, 22.68, 25.90, 29.35, 29.61,29.64, 29.68, 29.69, 31.91, 34.27, 54.79, 57.26, 73.65, 2.89, 4.25,4.77, 140.17; FABMS m/z 465 (M+H), 448 (M-H₂O), 464 (M−H), 388 (M-Ph),224 (M-C₁₆H₃₃O); HRFABMS calcd for C₃₂H₅₂NO M_(r) 466.4049 (M+H), foundM_(r) 466.4037.

The assignment of the 2S,3R configuration is based on comparison of thechemical shifts of the benzyl protons in 60 to literature values for thesyn and anti diastereomers of 2-(N,N-dibenzylamino)-3-pentanol. The antiisomer has a chemical shift difference of 0.29 ppm. and the syn is 0.52ppm. Comparison of other syn-anti pairs show the range for the synisomer to be 0.44 to 0.54 ppm and the anti 0.05 to 0.29 ppm. The valuefor 60 is 0.29 ppm.

(2S,3R)-2-Amino-3-octadecanol (1)

To a 15 ml round bottom was added 60 (88.2 mg, 0.189 mmol) in MeOH (2ml) and 20% Pd (OH)₂—C (11 mg). The mixture was stirred under 1atmosphere of hydrogen overnight. The catalyst was removed by filtrationthrough a 25 mm syringe filter (0.2 μm nylon membrane) and the filterwas washed with 4 ml of MeOH. The solvent was then removed in vacuo togive 51.50 mg of a white solid. The product was purified bychromatography over a 6 ml LC-Si SPE tube (90:10 CH₂Cl₂/MeOH followed by100% MeOH) to give 49.47 mg (92% yield) of a white solid: mp 66–67° C.;[α]²⁶ _(D)=+24.9 (c 1, CHCl₃); ¹H NMR (500 MHz, CD₃OD) δ 0.89 (t, 3H,J=7.0 Hz), 1.05 (d, 3H, J=6.6 Hz), 1.20–1.56 (bm, 31H), 2.81 (qd, 1H,J₁=6.6 Hz, J₂=3.8 Hz), 3.42 (dt, 1H, J₁=8.8 Hz, J₂=3.8 Hz); ¹³C NMR (1Mhz) δ 14.60, 16.82, 23.90, 27.40, 30.65, 30.90, 30.95, 30.96, 33.23,34.13, 52.33, 76.16; FABMS m/z 286.3 (M+H), 268.3 (M-OH), HRFABMS calcdfor C₁₈H₄₀NO M_(r) 286.3110 (M+H), found M_(r) 286.3109.

A mixture of diastereomers of3-hydroxy-2-(1-methyl-2-2-hydroxy-heptadecyl)-isoindolin-1-one (152, 22mg) were separated by cyano HPLC with hexane/2-propanol (98:2, 1 ml/min)to give four compounds (152a–152d). The purity of each peak wasdetermined by reinjection on HPLC. Anal Calcd. For C₂₆H₄₄NO₃: 418.3321(M+H). Found: 418.3321 HRFABMS).

152a: 4.2 mg; t_(r) 13.3 min; ¹H NMR (CDCl₃) δ 7.77 (1H, d, 7.3), 7.58(2H, m), 7.50 (1H, m), 5.91 (2H, s), 4.51 (1H, m), 3.78 (1H, m), 1.58(2H, m), 1.40 (3H, d, 7.1), 1.24 (26H, m), 0.87 (3H, t, 6.5); FABMS m/z418, 400; relative ratio of diastereomers 17:1:0:0 (152a: 152b: 152c:152d).

152b: 13.7 mg; t_(r) 13.9 min; ¹H NMR (CDCl₃) δ 7.70 (1H, d, 7.3), 7.54(2H, m), 7.47 (1H, m), 5.88 (2H, s), 4.37 (1H, m), 3.85 (1H, m), 1.52(2H, m), 1.27 (3H, d, 7), 1.25 (26H, m), 0.87 (3H, t, 6.5); FABMS m/z41, 400; relative ratio of diastereomers 1:6.8:0:0(152a:152b:152c:152d).

152c: 1.4 mg; t_(r) 20.0 min; ¹H NMR (CDCl₃) δ 7.78 (1H, d, 7.3), 7.59(2H, m), 7.51 (1H, m), 5.93 (2H, s), 4.12 (1H, m), 3.99 (1H, m), 1.58(2H, m) 1.37 (3H, d, 7.0), 1.25 (26H, m), 0.87 (3H, t, 6.5); FABMS m/z418, 400; relative ratio of diastereomers 0:2.5:45:1(152a:152b:152c:152d).

152d: 1.5 mg; t_(r) 21.7 min; ¹H NMR (CDCl₃) δ 7.77 (1H, d, 7.3), 7.59(2H, m), 7.51 (1H, m), 5.86 (2H, s), 4.12 (1H, m), 3.90 (1H, m), 1.58(2H, m), 1.45 (3H, d, 6.6), 1.24 (26H, m), 0.87 (3H, t, 6.5); FABMS m/z418, 400; relative ratio of diastereomers 0:1:2:21(152a:152b:152c:152d).

Each diastereomer was separately deprotected by the method of Osby etal. Each isomer was dissolved in 2-propanol/water (6:1, 0.1 M for 152aand 152b, 0.7 M for 152c and 152d). Sodium borohydride (5–10equivalents) was added to each solution, which was then stirred at 25°C. for 24 h. Each solution was then adjusted to pH 4.5 with acetic acidand stirred at 80° C. for an additional 24 h. Ammonium formate was addedto bring the pH of each solution to above 7 and then the solvent wasremoved from each by a stream of nitrogen. The residue from each wasapplied to a silica SPE column, which was first washed withhexane:2-propanol (9:1) and then the product eluted with 2-propanol. ¹HNMR indicated that 152a and 152d produced 154 (1.15 mg, 40%, and 0.48mg, 47% respectively), while 152b and 152c produced 155 (3.35 mg, 42%,and 0.38 mg, 40%, respectively).

154: White solid; silica TLC (3:12:2:2 CHCl₃/1-BuOH/AcOH/H₂O) R_(f) 0.48(ninhydrin-positive, pink); IR (NaCl) 2919, 2851, 1563, 1466, 1406, 758cm⁻¹; FABMS m/z 438, 286, 268, 85, 70, 69, 57, 55, 44. Anal. Calcd. ForC₁₈H₄₀NO: 286.3110 (M+H). Found 286.3115 (HRFABMS).

155: White solid; silica TLC (3:12:2:2 CHCl₃/1-BuOH/AcOH/H₂O) R_(f) 0.50(ninhydrin-positive, pink); IR (NaCl) 3281, 2917, 2849, 1568, 1520,1470, 1412 cm⁻¹; FABMS m/z 438, 286, 268, 85, 70, 69, 57, 55, 44, Anal.Calcd. For C₁₈H₄₀NO: 286.3110 (M+H). Found: 286.3109 (HRFABMS).

Acetylation

A portion of fraction B (560 μg) dissolved in acetic anhydride (200 μL)and pyridine (400 μL) and was stirred at 25° C. for 4.5 h, at which timeno starting material could be observed by TLC. The solvent was removedby a stream of nitrogen to give AcB: off-white solid; silica TLC R_(f)0.86 (3.12:2:2 CHCl₃/1-BuOH/AcOH/H₂O, phosphomolybdic acid), 0.65 (9:1CHCl₃/MeOH, phosphomolybdic acid); IR (NaCl) 2922, 2853, 1741, 1651,1547, 1460 1371, 1234, 1022, 970 cm⁻¹; FABMS m/z 370, 310, 268; CIMS m/z426, 424, 412, 410, 398, 384, 370, 368, 364, 338, 324, 310, 165, 149,139, 1, 121, 111, 97, 86, 61, 57, 55. Anal. Calcd. For C₂₂H₄₄NO₃:370.3321 (M+H). Found: 370.3326 (HRFABMS).

Triacetylsphingosine (133)

In a procedure similar to Grode and Cardellina D-erythro-sphingosine (4,2 mg, 6.7 μmol, Sigma) in acetic anhydride (1 ml) and pyridine (2 ml)was stirred at 25° C. for 4.5 h, at which time no starting materialcould be observed by TLC. The solvent was removed by a stream ofnitrogen to give 133: white solid; silica TLC R_(f) 0.86 (3:12:2:2CHCl₃/1-BuOH/AcOH/H₂O, phosphomolybdic acid), 0.65 (9:1 CHCl₃/MeOH,phosphomolybdic acid); FABMS m/z 580, 426, 366, 306, 264; CIMS m/z 468,454, 426, 424, 394, 366, 364, 306, 264, 144, 85, 84, 83, 61.

(2S,3S)-2-Acetamido-3-acetoxyoctadecane (156)

(2S,3S)-2-amino-3-octadecanol (154, 150 μg, 0.5 μmol) in aceticanhydride (50 μl) and pyridine (100 μl) was stirred at 25° C. for 5 h,at which time no starting material could be observed by TLC. The solventwas removed by a stream of nitrogen to give 156: white solid; silica TLCR_(f) 0.86 (3:12:2:2 CHCl₃/1-BuOH/AcOH/H₂O, phosphomolybdic acid); IR(NaCl) 3286, 2924, 2853, 1740, 1653, 1541, 1456, 1371, 1238 cm⁻¹; FABMSm/z 522, 370, 328, 310, 286, 268. Anal. Calcd. For C₂₂H₄₄NO₃: 370.3321(M+H). Found: 370.3326 (HRFABMS).

(2S,3R)-2-Acetamido-3-acetoxyoctadecane (157)

(2S,3R)-2-amino-3-octadecanol (155, 750 μg, 2.6 μmol) in aceticanhydride (200 μL) and pyridine (400 μL) was stirred at 25° C. for 5 h,at which time no starting material could be observed by TLC. The solventwas removed by a stream of nitrogen to give 157: white solid; silica TLCR_(f) 0.86 (3:12:2:2 CHCl₃/1-BuOH/AcOH/H₂O, phosphomolybdic acid); IR(NaCl) 3289, 2917, 2849, 1728, 1637, 1545, 1464, 1369, 1240 cm⁻¹; FABMSm/z 522, 370, 328, 310, 286, 268. Anal. Calcd. For C₂₂H₄₄NO₃: 370.3321(M+H). Found: 370.3319 (HRFABMS).

Spisulosine 285 Acetonide (146)

A portion of fraction A (40 μg) was dissolved in acetone (200 μL) towhich 0.1 N hydrochloric acid (20 μL) was added. This solution wasstirred at 25° C. for 24 h, after which the solvent was removed by astream of nitrogen. FABMS indicated that a small amount of the acetonide146 was formed: m/z 592, 452, 438, 326.3430, 300, 286, 268. Anal. Calcd.For C₂₁H₄₄NO: 326.3423 (M+H). Found 326.3430 (HRFABMS).

(4S,5R)-4-Methyl-5-(n-pentadecyl)-oxazolidinone (158)

(2S,3R)-2-amino-3-octadecanol (155, 750 μg, 2.6 μmol) was dissolved indichloromethane (100 μL) to which 1; 1′-carbonyldiimidazole (0.85 mg,5.3 μmol) and triethylamine (0.4 μL, 2.9 μmol) was added. The solutionwas stirred for 5 h and then the solvent removed by a stream ofnitrogen. The crude product 158 was analyzed without purification: IR(NaCl) 36, 2919, 2851, 1742, 1713, 1551, 1470, 1395, 1321, 49, 1239,1094, 1061, 1001, 768, 743, 664 cm⁻¹; FABMS m/z 785, 623, 474, 406, 362,328, 312, 286, 268. Anal. Calcd. For C₁₉H₃₈NO₂: 312.2903 (M+H). Found:312.2903 (HRFABMS).

Further Investigation of Changes in Cell Morphology

Materials

Lysophosphatidic acid (LPA), antibodies against tubulin and phalloidinwere all obtained from Sigma. Fluorescein- and Texas red-labelled goatantimouse antibody were obtained from Amersham (U. K.). Antibody raisedagainst the Rho protein was obtained from Sta Cruz Biotechn.

Cell Culture

Vero cells were grown in Dulbecco's modified Eagle medium supplementedwith 10% foetal bovine serum. Spisulosine or LPA were added to thesecultures to a concentration of 0.2–1.0 mg and 50–10 mM respectively,from 4 to 24 hours. Cells were counted with the drug exclusionhaemocytometer procedure using a solution of 0.4% Trypan blue in Hanksbuffered Saline (Celis and Celis, “General Procedures for Tissue Culturein Cell Biology, a Laboratory Handbook” Academic Press Inc, Vol 1, pp.5–17.)

EXAMPLE A Spisulosine 285 Causes Changes in Cell Morphology

Vero cells were incubated with spisulosine 285 (0.5 mM) for 4 hours.FIG. 3 is a microphotograph for the results in Example A. Cell shapedwas altered from polygonal (untreated cells, panel a) to a fusiformshape (panel b). Panel c represents a higher magnification of theculture to which spisulosine was added.

EXAMPLE B Change in Cell Morphology is Due to an Effect on the CellMicrofilaments

In order to identify the organization of the microfilament andmicrotubule organization in cells treated with spisulosine 285, cellswere stained with phalloidin to detect actin polymers, and anantitubulin antibody to detect tubulin.

Vero cells were incubated in the presence (panel b, d) or absence (a, c)of 0.5 mM spisulosine 285 for 4 hours. Cells grown in coverslips werefixed with methanol at −20° C. (for tubulin antibody) or with 4%paraformaldehyde in phosphate buffered saline PBS (w/v) for phalloidinincubation. In the second case the cells were washed with 0.2% TritonX100 in PBS. The coverslips were washed with PBS and incubated for 1hour at room temperature with the tubulin antibody (diluted 1/1000 inPBS) or with phalloidin (1 mg/ml). After washing with PBS the coverslipsincubated with the tubulin antibody were overlaid with fluorescein orTexas red-labelled goat antimouse antibodies (diluted 1:50 in PBS). Thecoverslips were mounted with Mowiol and stored in the dark at 4° C.until observation.

FIG. 4 is a microphotograph for the results in Example B. Panel ‘a’represents cells stained with phalloidin (actin stain) and not treatedwith spisulosine. Panel ‘b’ represents cells stained for phalloidin andtreated with spisulosine. Panel ‘c’ represents cells stained for tubulinand not treated with spisulosine. Panel ‘d’ represents cells stained forphalloidin and treated with spisulosine. There is a dramatic decrease inactin in spisulosine-treated cells, in comparison with untreated cells.Under the same conditions, the microtubule network remains in apolymerised form.

EXAMPLE C Effect of Spisulosine 285 on the Rho Protein

The small GTP binding protein Rho is involved in the formation ofactin-myosin “stress fibers” (Hall, A., Science, 279, 1998, p 509–514).Therefore, the electrophoretic mobility and cellular distribution of Rhowas analyzed in cells treated with spisulosine 285.

FIG. 5 is an electrophoretogram of Example C. In panel A, equivalentamounts of protein from a cell extract from untreated (a) or from 0.5 mMspisulosine 285 treated (20 hour) cells (b) were fractionated by gelelectrophoresis and blotted onto nitrocellulose paper to analyze theamount of the Rho protein.

In panel B the experiment was carried out as above, except that thehomogenate was fractionated into a particulate (membrane, “M”) fractionand soluble (“S”) fraction.

Subcellular fractionation was carried out by placing cells in ahypotonic buffer (0.25 M sucrose, 20 mM HEPES pH 7.4, 2 mM EDTA, 1 mMPMSF, 10 mg/ml aprotinin, leupeptine and pestatine), and lysing themwith a Dounce. The homogenate was first centrifuged at 750 g for 5minutes to remove nuclei and unbroken cells, and the supernatant wasfurther centrifuged at 30,000 g for 1 hour (4° C.) to isolate a pelletedparticulate fraction (putative membrane fraction) and a supernatant. Thedifferent fractions were characterised by electrophoresis and WesternBlotting using an antibody against the Rho protein.

No significant change in the amount or mobility of Rho was observed ontreatment of cells with spisulosine 285. However, a decrease in theproportion of Rho associated with the particulate fraction was observed.

EXAMPLE D Effect of Lysophosphatidic Acid (LPA) on the Action ofSpisulosine 285

LPA is known to increase the level of stress fibers in cells byactivation of the Rho protein. The effect of LPA on cells treated withspisulosine 285, and untreated cells was examined.

Vero cells were incubated in the absence (a) or presence (b) of 10 mMLPA for 2 hours, or in the presence (c) of 0.5 mM spisulosine 285 for 20hours, or in the presence (d) of first 10 mM LPA (2 hours) andafterwards with 0.5 mM spisulosine 285 for an additional 18 hours.

FIG. 6 is a microphotograph for the results in Example D. Panel bindicates the effect of LPA in increasing the level of actin. Incubationof Vero cells with spisulosine for 24 hours results in the appearance ofrounded cells, see panel c. These cells detach from the culture dish anddie. The addition of LPA prior to spisulosine prevents the morphologicalchange promoted by spisulosine.

In Vivo Data

EXAMPLE E The Effect of Spisulosine 285 In Vivo

Spisulosine 285 was tested in in vivo studies against xenograft modelsof human prostate cancer (PC-3) and human renal cancer (MRI-H-121).These models use subcutaneously implanted solid human tumors that growand increase in volume over time. The mean volume of tumor growth incontrol animals provides the basis for comparison. For active compoundsthe tumor growth is inhibited either completely (% T/C values<1%, ornegative), or partially (>1% T/C−50% T/C). A level of activity that isless than 40% T/C is considered statistically significant. The doses ofspisulosine used were given at the maximum tolerated, non lethal dose(MTD), ½ MTD and ¼ MTD. Delivery of the drug was by the intraperitonealroute.

Human prostate cancer PC-3 Total Dose % Compound (mg/kg) T/C DayComments Spisulosine 285 9.990 −21% 11 stasis (complete remission)Spisulosine 285 5.010  −1% 11 stasis (complete remission) Spisulosine285 2.499 223% 15 Control 100% 15

Human MRI-H-121 renal cancer Total Dose Compound (mg/kg) % T/C DayComments Spisulosine 285 9.990 28% 11 inhibition (partial remission)Spisulosine 285 5.010 35% 11 inhibition (partial remission) Spisulosine285 2.499 43% 15 Control 100%  15

Spisulosine 285 is effective against both tumour types, significantlyreducing the tumour size in the case of the human prostate cancer modelPC-3 at higher doses. Spisulosine 285 reduces the growth of the humanrenal cancer, with effects continuing up to a few weeks after the lastdose of the drug.

EXAMPLE F

An expanded in vitro screen was performed of spisulosine 285 against aseries of different cell lines. The following data was obtained:

CV-1 Therapeutic Category Line Tumor IC50 Index Solid SK-HEP-1 Liver3.51E−15 7863 PANC-1 Pancreas 1.71E−12 16 HT-29 Colon 2.56E−12 11 786-0Renal 2.75E−12 10 FADU Pharnynx 4.99E−12 6 Hs 746T Stomach 7.89E−12 3SK-OV-3 Ovary 1.40E−11 2 MX-1 Mammary 3.89E−11 1 RAMOS Burkitts 4.82E−111 P3HR1 Burkitts 6.73E−11 0 SW684 Fibrosarcoma 1.05E−09 0 Lymphoma U-937Lymphoma 1.96E−11 1 H9 Lymphoma 3.10E−11 1 Leukemia HL60 Leukemia8.50E−12 3 ARH77 Leukemia 1.36E−12 2 K562 Leukemia 1.57E−11 2 CCRF-SBLeukemia 1.05E−09 0 Normal CV-1 Kidney 2.76E−11 1 fibroblasts

The range of IC₅₀ potencies against the tumor cell lines are fromnanomolar, 1.05 E-09 nM, to femtomolar, 3.51 E-15 mM. It is exceptionalto go beyond the nM and pM range to find a drug which has activity inthe fM range.

The activities against the solid tumours were generally 1 log morepotent than against the leukemias and lymphomas. Among the solidtumours, the most slow growing were the most sensitive, culminating withthe very slow growing hepatoma SK-HEP-1.

The best therapeutic indices compared to the CV-1 normal cell line wereseen with the slow growing solid tumors, since the IC₅₀ potency (2.76E-11) was comparable to the leukemia/lymphomas. The solid tumor TIsranged from 1–20 units and the TI for the hepatoma was >3 log.

The renal tumour cell line was in the most active group, pM potencies,which correlates well to the in vivo xenograft data.

REFERENCES

The following references provide background information related to thepresent invention. The disclosures of each are hereby incorporatedherein by reference.

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The present invention has been described in detail, including thepreferred embodiments thereof. However, it will be appreciated thatthose skilled in the art, upon consideration of the present disclosure,may make modifications and/or improvements on this invention.

1. A pharmaceutical composition comprising an isolated and purifiedlong-chain, straight-chain 2-amino-3-hydroxyalkane, or prodrug thereof,and a pharmaceutically acceptable carrier, wherein the carbon chain inthe long-chain, straight-chain 2-amino-3-hydroxyalkane is C₁₆–C₂₄. 2.The composition according to claim 1, wherein the carbon chain in thelong-chain, straight-chain 2-amino-3-hydroxyalkane is C₁₈–C₂₀.
 3. Thecomposition according to claim 2, wherein the carbon chain in thelong-chain, straight-chain 2-amino-3-hydroxyalkane is C₁₈.
 4. Thecomposition according to claim 1, wherein the carbon chain in thelong-chain, straight-chain 2-amino-3-hydroxyalkane is C₁₆.
 5. Thecomposition according to claim 1, wherein the carbon chain in thelong-chain, straight-chain 2-amino-3-hydroxyalkane is C₂₀–C₂₄.
 6. Thecomposition according to claim 1, wherein the pharmaceuticallyacceptable carrier is a solution compatible with cells.
 7. Thecomposition according to claim 1, wherein the long-chain, straight-chain2-amino-3-hydroxyalkane is a compound of Formula I:

where n=12, 13, or
 14. 8. The composition according to any one of claims1–7, further comprising an additional drug for combination cancertherapy.
 9. A method of preparing a pharmaceutical composition for thetreatment of cancer, wherein the method comprises the step of combininga long-chain, straight-chain 2-amino-3-hydroxyalkane or prodrug thereofwith a pharmaceutically acceptable carrier and optionally an additionaldrug for combination cancer therapy, wherein the carbon chain in thelong-chain, straight-chain 2-amino-3-hydroxyalkane is C₁₆–C₂₄.
 10. Amethod according to claim 9, wherein the long-chain, straight-chain2-amino-3-hydroxyalkane is a compound of Formula I:

where n=12.